Staphylococcus aureus is a human pathogen that can cause serous illnesses. The organism is constantly evolving and strains that are more virulent and more resistant to antibiotics have emerged in recent years. As a result, the infections caused by this organism have become increasingly difficult to treat. New methods of treatment are therefore urgently needed. S. aureus pathogenicity depends on a plethora of virulence factors produced by the organism, which are regulated by an equally impressive number of regulators in a surprisingly complex manner that is far from been understood. Understanding virulence regulation is the key to understand pathogenesis, which is needed for providing fundamental knowledge for developing new methods of treatment. Most strains of S. aureus produce one type of capsular polysaccharide that is either type 5 or type 8. Because of their predominance these capsules have been the prime components for vaccine development. Recent animal studies using various models have unequivocally shown that type 5 and type 8 capsules play an important role as an antiphagocytic factor in staphylococcal pathogenesis. However, like other virulence factors, little is known about the regulation of these capsules especially in vivo. Recently, we have identified and characterized several regulators affecting capsule production. Our studies indicate that type 5 and 8 capsules are regulated by a larger number of regulators most of which also regulate other virulence factors. Based on these results, we hypothesize that capsule is highly regulated by a complex regulatory network in responding to various environments that the bacteria encounter during pathogenesis. It is worth noting that some of these regulators are uncommon as a transcriptional regulator suggesting an uncommon mode of regulation may be involved. Accordingly, in this proposal, we will focus on elucidating capsule regulatory mechanisms in vitro and in vivo as a model to understand virulence gene regulation in S. aureus. We propose to accomplish three specific aims: (i) to characterize the regulatory network affecting capsule production; (ii) to elucidate the mechanisms of regulation; (iii) to investigate the regulation of capsule in vivo and to relate virulence gene regulation to pathogenesis. The successful completion of the studies outlined in this application not only will provide further insight on capsule regulation but also will contribute to our understanding of the overall mechanism of virulence gene regulation. The new knowledge gained from these studies will provide a firm basis for developing novel methods for controlling staphylococcal infections.